1. Field of the Invention
The present disclosure generally relates to liquid chromatography. More particularly, the disclosure relates to detectors configured to measure the bulk refractive index of solutions used in connection with liquid chromatography procedures.
2. Description of the Related Art
Refractive index detectors in high performance liquid chromatography (HPLC) garnered use as a means to analyze samples that lacked strong chromophores in ultraviolet or visible regions, and that were non-fluorescent. The most notable advantages of refractive index detection are: (1) the technique is non-destructive enabling downstream analysis, and (2) refractive index is a concentration dependent bulk property classifying it is as an universal detector (i.e., all compounds with polarizable electrons can be detected under proper conditions). The differential refractometer was one of the earliest implementations of refractive index detection in liquid chromatography and the design has remained popular over the past half-century. The setup contains a sample and a reference flow cell with the cell temperature tightly maintained with a thermostat, commonly at 30° C. or greater.
Comparing changes in bulk refractive index sample versus reference cells of identical mobile phase and temperature enabled sensitivities as low as 10−7 RIU (refractive index units) and detection limits of approximately 0.1% sample by volume.
However, the reference cell is also the source of major limitations of RI detection for liquid chromatography separations including: (1) the inability to perform gradient separations and (2) extended down time to allow for thermal equilibration of flow cells. Consequently, the limited sensitivity, small dynamic range, and extended equilibration times have limited the use of the differential refractometer and refractive index detection in liquid chromatography to specialty applications, such as lipid, sugar, and protein detection for food analysis.
In the decades following the introduction of refractive index sensing to liquid chromatography, there were multiple attempts to improve upon the original design to increase its applicability. Notable modifications include the elimination of the need to operate at elevated temperature, incorporation of a second column and pumping system enabling analysis of gradient separations, continuous alteration of the laser interrogation angle to adjust for refractive index changes in mobile phase composition to allow gradient separations, and the use of thermooptic, interferometric, and liquid core optical ring resonator (LCORR) methods to increase sensitivity.
However, the design modifications were insufficient to justify significant commercialization and industry adoption, as each improvement only offered a solution to one of the major limitations of refractive index detection.